The surface glycoproteins of enveloped viruses act as anchors for docking and fusion with the target host membrane and initiation of another round of viral replication (Wyatt et al., Science, 280(5371): 1884-8 (1998)). These glycoproteins are the most prominent viral surface features that can be recognized within the host cellular background and targeted for antibody neutralization. Consequently, viruses have evolved a number of strategies for shielding the spike structures formed by their glycoproteins. These strategies include restriction of access to conserved structural features through conformational occlusion and oligomerization (Kwong et al., Nature, 420(6916): 678-82 (2002); and Labrijn et al., J. Virol., 77(19): 10557-65 (2003)), sequence hyper-variability especially within loops that mask conserved epitopes (Starcich et al., Cell, 45(5): 637-48 (1986)), and extensive posttranslational glycosylation (Wei et al., Nature, 422(6929): 307-312 (2003)). In case of HIV, the viral defenses appear to be further buttressed by limiting the number of gp120 trimeric spikes present on the HIV envelope and effectively reducing the quantity of viral antigen presented to the immune system (Klein et al., PLoS Pathog., 6(5): e1000908 (2010)).
N-linked carbohydrates compose approximately 50% of the molecular weight HIV gp120 (Leonard et al., J. Biol. Chem., 265(18): 10373-82 (1990)), creating a glycan armor that hides the underlying protein structures. The success of this defensive mechanism may in part hinge on the weak interactions between proteins and carbohydrates (Toone, Current Opinion in Structural Biology, 4(5): 719-728 (1994)). The viral glycan modifications are essential for proper folding and trafficking of viral glycoproteins within the endoplasmic reticulum and trans-Golgi network. Therefore, potential mutations that would reduce the glycosylation levels of HIV glycoproteins could affect processing and maturation of these glycoproteins, leading to attenuated infectivity, as well as exposing the virus to the immune system.
Lectins are small proteins that have evolved to bind carbohydrates with high affinity and specificity. A number of lectins have been shown to display potent antiviral activity (Balzarini et al., Antivir. Chem. Chemother., 18(1): 1-11 (2007)). A potent anti-HIV lectin is griffithsin (GRFT), an obligate domain-swapped dimer in which each domain has jacalin-like fold (Bourne et al., Biochem. J., 364(Pt. 1): 173-80 (2002)). Unlike jacalin in which only a single carbohydrate-binding site is present in each molecule (Jeyaprakash et al., J. Mol. Biol., 332(1): 217-28 (2003)), each domain of GRFT contains three carbohydrate-binding sites, the centers of which form an equilateral 15-A triangle (Ziolkowska et al., Structure, 14(7): 1127-35 (2006)). GRFT has anti-HIV EC50 of ˜50 pM in cell-based assays (Mori et al., J. Biol. Chem., 280(10): 9345-53 (2005)). GRFT is also active against the coronavirus responsible for SARS (O'Keefe et al., J. Virol., 84(5): 2511-21 (2010); and Zeitlin et al., Proc. Natl. Acad. Sci. USA, 106(15): 6029-30 (2009)) and against the hepatitis C virus (Meuleman et al., Antimicrob. Agents Chemother., 55(11): 5159-67 (2011)).
GRFT is thermostable, can survive in a wide range of conditions including macaque vaginal environment, and exhibits little or no toxicity and immunogenicity (Kouokam et al., PLoS One, 6(8): e22635 (2011)). Large-scale production of GRFT in genetically modified tobacco plants has been demonstrated (O'Keefe et al., Proc. Natl. Acad. Sci. USA, 106(15): 6099-104 (2009)).
The structures of unliganded, native GRFT and its complexes with a number of mono- and disaccharides have been previously identified (Ziolkowska et al., Structure, 14(7): 1127-35 (2006); Ziolkowska et al., Acta Biochim. Pol., 53(4): 617-26 (2006); and Ziolkowska et al., Protein Sci., 16(7): 1485-9 (2007)). Additionally, several monomeric forms of GRFT (mGRFT) have been engineered and their structures solved, including a complex with nonamannoside, an analogue of Man9 and a common glycosylation pattern found on HIV surface glycoproteins. Although, the anti-HIV activity of mGRFT was approximately 1000-fold lower than that of GRFT, both the monomeric and dimeric forms of this lectin have very similar carbohydrate binding affinities.
The need remains for additional griffithsin forms with improved potency.